Synthetic antisense or siRNA (small interfering) oligonucleotides as gene silencing agents inhibit viral replication and expression of disease-causing genes based on the simple concept of nucleic acid sequence recognition via Watson-Crick hydrogen bonding by a complementary base sequence. The 2′-O-methoxyethyl substituted antisense oligonucleotides (MOE-AONs) are currently being studied in several ongoing clinical trials and have shown excellent safety profiles. The success of MOE-AONs was attributed to the gauche interactions of vicinal 2′-O— and the methoxy-substitution, thereby imparting favorable preorganization, favoring the hydration of DNA:RNA duplex minor groove and protecting against hydrolytic cleavage due to steric hindrance in the minor groove. In spite of the high thermal stability of the duplexes containing MOE-AONs (ΔTm≈1° C./MOE unit), a large difference between their IC50 and the ED50 values was observed. The reason for this difference could be primarily arising from degradation of the antisense oligonucleotides (AONs) under physiological conditions and it is evident that it is of interest to reduce this difference and to optimize the therapeutic value of MOE-AONs. This should indeed be achievable by blocking the hydrolytic active site of the phosphodiesterase enzymes without compensating the target recognition ability of MOE-AONs. Antisense drugs are being researched to treat cancers (including lung cancer, colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such as asthma, arthritis and pouchitis with an inflammatory component.
U.S. Pat. No. 4,503,252 discloses the method for the synthesis of serinol comprising reacting 1,3-dimethoxy-isopropylchloride with excess ammonia by heating to form 1,3-dimethoxy-isopropylamine and subsequently converting this intermediate to serinol by refluxing it with aqueous hydrochloric acid. Selective alkylation of uridine at 2′-position such as treatment of uridine with alkyl halides using DBTO or by employing 3′,5′-O- and N-protected uridine in reactions with alkyl halides reported by Martin, P. in Helv. Chim. Acta, 1995, 78, 486. Saneyoshi, H in M. J. Org. Chem. 2005, 70, 10453 describes the reaction of appropriately protected ribonucleoside derivatives with acrylonitrile in t-BuOH in the presence of Cs2CO3 gave 2′-O-cyanoethylated ribonucleoside derivatives in excellent yields, which were converted by a successive selective deprotection/protection strategy to 2′-O-cyanoethylated 5′-O-dimethoxytritylribonucleoside 3′-phosphoramidite derivatives in high yields. In addition, it provides that introduction of a cyanoethyl group into the 2′-position of RNA resulted in significant increase of nuclease resistance toward snake venom and bovine spleen phosphodiesterases. Additionally, Legorburu, U in Tetrahedron, 1999, 55, 5635 describes conversion of uridine into 2′-O-(2-methoxyethyl) uridine and 2′-O-(2-methoxyethyl) cytidine. Manoharan, M. et al. J. Org. Chem. 1999, 64, 6468 reported N-(2-Cyanoethoxycarbonyloxy) succinimide as new reagent for protection of amino groups in oligonucleotides, Lecubin F. in journal of Nucleosides Nucleotides Nucleic Acids, 2003 May-August; 22(5-8): 1281-4 discloses synthesis and triplex binding properties of oligonucleotides containing a novel nucleobase, whereas triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair. Further Yuichi Nakamura in Org. Lett., 2013, 15 (2), pp 322-325 Publication: Dec. 26, 2012 describes siladenoserinols a-l as sulfonated serinol derivatives from a tunicate as inhibitors of p53-hdm2 interaction, a promising target for cancer chemotherapy.
Recently, there have been some fine efforts in this direction to combine the structural aspects of locked nucleic acids (LNA) with MOE-AONs (S/R-cMOE, S/R cEt). Indeed, these additional modifications stabilize modified LNA against enzymatic cleavage improving their potency mainly by AONs stabilization to potential in vivo degradation. It was further shown by crystal structural data for the duplex containing a single unit of cMOE that the constrained LNA framework, substituted with a —CH2OMe or Me groups, brings these substitutions close to the phosphate, preventing, for steric reasons, the metal binding required for the hydrolytic cleavage. Furthermore, the efficiency of both cMOE diastereoisomeric derivatives is similar in blocking the enzyme activity. The cost of LNAs, together with the additional synthetic steps in the preparation of functionalized LNA derivatives, probably prevents their use as therapeutic agents. LNA is also known to show hepatotoxicity. The stability of MOE and LNA analogs to hydrolytic enzymes is low, and requires phoshorothioates for further stabilization against enzymatic degradation.
The synthetic approach for modifications at 3′- and 5′-end as well as at 2′-position is easy and scalable as compared to the other alternatives such as combining LNA and MOE strategies. The LNA/MOE combination leads to cMOE and cEt modifications do stop degradation of oligomers but without chiral discrimination, completely depending upon steric bulk of bicylic system. The above synthesis further involves several steps and separation of diastereomers.
To overcome the above drawbacks, the present inventors have synthesized new serinyl compounds characterized by the presence of a positively charged amino functionality in conjunction with the methoxy substituent on the 2′-O-alkyl chain, in particular, due to electrostatic repulsions, a positively charged amino functionality may potentially displace the metal ion, required for the hydrolytic cleavage. Further, the stereospecific addition of protonable amino group to the 2′-O-methoxyalkyl component preferentially resists hydrolytic cleavage by enzymes without use of phosphorothioate. Advantageously the presence of an ammonium group on modified MOEs improves the hydration in the minor groove enhancing the stabilization of the duplex.
Therefore the present inventors have prepared novel 2′/3′/5′-R/S serinyl functionalized nucleic acids to save ODNs (oligodeoxyribonucleotides) from degradation without derivatization at phosphorus; and also to make better recognition strength of oligonucleotides, because the phosphorothioate analogs incur loss in binding strength to target RNA.